Biodegradable polymer/hydrogel composite for controlled delivery of cationic formulations

Composites of biodegradable polymers and hydrogels are promising materials for controlled delivery systems with prolonged drug release. In this contribution, we present an innovative implant design comprising poly(DL-lactide-co-ε-caprolactone) copolymer base and a crosslinked poly(acrylic acid) hydrogel. Implants were prepared in the form of disks using the modified traditional liquid phase inversion process. Solutions containing all implant precursors were dispensed into transparent non-stick molds and cured by UV irradiation. UV curing was followed by immersion into the phosphate buffer solution bath to achieve phase separation and solidification. Structure and composition of the implant were characterized using SEM and FTIR. Obtained implants exhibited high loading capacity for cationic formulations and a moderate degree of swelling. Studies of implant loading and subsequent release of methylene blue into the phosphate-buffered saline demonstrated diffusioncontrolled delivery kinetics over a period of several weeks. To assess biocompatibility of implants as possible materials for drug delivery systems in mammals, we evaluated their effects on viability (Trypan blue exclusion assay), metabolic activity, proliferation (MTT assay) and priming (nitric oxide/NO production) of freshly isolated rat splenocytes during 24 h and 48 h of cultivation. The viability was unaltered, metabolic activity/proliferation was increased after 48 h and the decrease of NO production, as well as drop in responsiveness to cell mitogen concanavalin A (ConA) in cells on implants were observed. These results suggest that implants could be used as a suitable material for drug delivery systems, but their capacity to stimulate cell proliferation and their immunosuppressive potential deserve further investigations

Sadržaj mikroelemenata u populacijama divljih vrsta suncokreta i hibrida

The aim of this work was to investigate genetic specificity of sunflower nutrition with microelements. Therefore, concentrations of essential (Zn, B, Mn, Cu, Fe and Ni) and non-essential (Cr, Al, Cd, As, Pb and Ba) micronutrients were analyzed. Five sunflower hybrids the most grown in Serbia and different populations of wild sunflower species originating from North America: Helianthus neglectus Heiser (3), Helianthus agrophyllus T&G (3), Helianthus petiolaris Nutt. (2), Helianthus annuus L. (4) were included in the experiment. Populations of wild sunflower species and hybrids differed significantly with respect to the concentration of analyzed elements. Manganese concentration was significantly higher in hybrids than in wild species. In all genotypes Fe, B and Mn had the highest concentration. Coefficient of variation of microelement concentration depended on genotype and particular element. In wild populations, for essential microelements, it was between 3.7 and 59.5, whereas in hybrids it varied from 10.0 to 48.8. Coefficient of variation of concentration of non-essential microelements in wild populations varied from 7.7 to 73.8, and in hybrids from 15.1 to 48.8. Average coefficient of variation in both wild species and hybrids was the lowest for Mn and Pb. It was the highest for Cr, Ni, and Zn in hybrids and for Cd, Ni, and Cr in wild species. The results suggest that genetic specificity with respect to uptake of microelements in wild species and hybrids is highly expressed. Broad genetic variability of concentrations of microelements in wild species and hybrids indicate that their reactions to deficiency and/or excess of those elements probably are not the same either. This finding may be used in breeding process aimed specifically at improvement of tolerance and capacity to accumulate microelements in sunflower. Phytoremediation technology designed to reduce the amount of microelements in the soil could thus be advanced by utilization of such plants.U cilju proučavanja genetske specifičnosti ishrane suncokreta mikroelementima ispitan je sadržaj neophodnih (Zn, B, Mn, Cu, Fe i Ni) i ne neophodnih mikroelemenata (Cr, Al, Cd, As Pb i Ba) u pet najrasprostranjenijih hibrida suncokreta u Srbiji kao i u različitim populacijama divljih vrsta suncokreta poreklom iz Severne Amerike: Helianthus neglectus Heiser (3), Helianthus agrophyllus T&G (3), Helianthus petiolaris Nutt. (2), Helianthus annuus L. (4). Ispitivane populacije divljih vrsta suncokreta i hibrida značajno su se međusobno razlikovale u pogledu sadržaja ispitivanih mikroelemenata. Najveća razlika između populacija divljih vrsta suncokreta i hibrida utvrđena je u sadržaju Mn koji je kod hibrida bio značajno veći. Kod svih ispitivanih genotipova najveći je bio sadržaj Fe, B i Mn. Koeficijent varijacije sadržaja mikroelemenata zavisio je od genotipa i elementa. Kod populacija divljih vrsta suncokreta kretao se od 3.7 do 59.5, a kod hibrida od 10.0 do 48.8. Za neesencijalne mikroelemente kod divljih populacija koeficijent je bio između 7.7, i 73.8, dok je kod hibrida varirao od 15.1 do 48.8. Koeficijent varijacije bio je najmanji kod Mn i Pb kod divljih vrsta i hibrida. Kod hibrida najveći koeficijent varijacije imali su Cr, Ni i Zn, a kod divljih vrsta Cd, Ni i Cr. Na osnovu dobijenih rezultata može se zaključiti da je genetička specifičnost u pogledu usvajanja neophodnih i drugih mikroelemenata kod populacija divljih vrsta suncokreta i hibrida veoma izražena. Široka genetska varijabilnost ispitivanih genotipova u pogledu sadržaja pojedinih mikroelemenata upućuje na pretpostavku o različitoj reakciji prema njihovom nedostatku i suvišku, što može da bude od značaja u oplemenjivačkom radu, posebno pri stvaranju genotipova podesnih za fitoremedijaciju zemljišta zagađenih mikroelementima

Sojne razlike u toksičnosti antagoniste vitamina K varfarina kod pacova

Warfarin (3-(alpha-acetonylbenzy1)-4-hydroxy coumarin) is a vitamin K (VK) antagonist that inhibits vitamin K-dependent (VKD) processes, such as blood coagulation. It also exerts an influence on some non-VKD-related activities. In this study, the effect of sub-acute (30-day) oral warfarin (2 and 1 mg L-1) intake on hematological parameters was examined in two rat strains, Albino Oxford (AO) and Dark Agouti (DA), that differ in their sensitivity to certain chemicals. Greater susceptibility to the anticoagulant effect of 2 mg L-1 of warfarin was observed in AO rats and was associated with an increase in the relevant hematological parameters in this strain. Although both strains responded to 2 mg L-1 of warfarin with quantitative changes in the peripheral blood leukocytes, differential bone marrow and lung responses were observed. Strain-related differences in the pro-inflammatory activity of peripheral blood granulocytes and in mononuclear cell IFN-gamma production were observed. Recognition of differences in quantitative and qualitative effects of oral warfarin on processes other than hemostasis might be of relevance for those humans who are on warfarin therapy.Varfarin (3-α-acetonilbenzil)-4–hidroksikumarin) je antagonist vitamina K (VK) koji inhibira procese zavisne od ovog vitamina, uključujući koagulaciju krvi. Osim toga, on ispoljava i aktivnosti koje ne zavise od vitamina K kao što su anti-tumorska i imunomodulatorna aktivnost. U ovom radu je ispitan efekat subakutnog (30 dana) oralnog unosa varfarina na hematološke parametre i aktivnost leukocita periferne krvi kod dva soja pacova Albino Oxford (AO) i Dark Agouti (DA) koji se raz- likuju u osetljivosti na iste hemijske agense. Kod jedinki AO soja zapažena je veća smrtnost nakon konzumiranja doze od 4 mg L–1 kao i veća osetljivost na antikoagulantno dejstvo varfarina pri nižim dozama (2 mg L–1) koje je praćeno povećanjem nekih hematoloških parametara. Iako kod jedinki oba soja dolazi do povećanja broja neutrofilnih leukocita periferne krvi pri dozi od 2 mg L–1, promene u osnovnim proinflamatornim aktivnostima ovih ćelija su zapažene samo kod jedinki DA soja. Promene u broju neutrofilnih leukocita u krvi DA jedinki su praćene povećanjem broja granulocitnih prekursora u koštanoj srži, dok prisustvo neutrofila u plućima AO jedinki ukazuje na razmenu ćelija između periferne krvi i plućnog intravaskularnog pula ćelija. Diferencijalne sojno–zavisne promene u aktivnosti mononuklearnih ćelija periferne krvi su takođe zapažene. Razlike u efektu oralno unetog varfarina mogu da imaju implikacije za osobe na oralnoj varfarinskoj terapiji

Strain differences in the toxicity of the vitamin K antagonist warfarin in rats

Warfarin (3-(α-acetonylbenzyl)-4-hydroxy coumarin) is a vitamin K (VK) antagonist that inhibits vitamin K-dependent (VKD) processes, such as blood coagulation. It also exerts an influence on some non-VKD-related activities. In this study, the effect of sub-acute (30-day) oral warfarin (2 and 1 mg L-1) intake on hematological parameters was examined in two rat strains, Albino Oxford (AO) and Dark Agouti (DA), that differ in their sensitivity to certain chemicals. Greater susceptibility to the anticoagulant effect of 2 mg L-1 of warfarin was observed in AO rats and was associated with an increase in the relevant hematological parameters in this strain. Although both strains responded to 2 mg L-1 of warfarin with quantitative changes in the peripheral blood leukocytes, differential bone marrow and lung responses were observed. Strain-related differences in the pro-inflammatory activity of peripheral blood granulocytes and in mononuclear cell IFN-γ production were observed. Recognition of differences in quantitative and qualitative effects of oral warfarin on processes other than hemostasis might be of relevance for those humans who are on warfarin therapy.Varfarin (3-α-acetonilbenzil)-4–hidroksikumarin) je antagonist vitamina K (VK) koji inhibira procese zavisne od ovog vitamina, uključujući koagulaciju krvi. Osim toga, on ispoljava i aktivnosti koje ne zavise od vitamina K kao što su anti-tumorska i imunomodulatorna aktivnost. U ovom radu je ispitan efekat subakutnog (30 dana) oralnog unosa varfarina na hematološke parametre i aktivnost leukocita periferne krvi kod dva soja pacova Albino Oxford (AO) i Dark Agouti (DA) koji se raz- likuju u osetljivosti na iste hemijske agense. Kod jedinki AO soja zapažena je veća smrtnost nakon konzumiranja doze od 4 mg L–1 kao i veća osetljivost na antikoagulantno dejstvo varfarina pri nižim dozama (2 mg L–1) koje je praćeno povećanjem nekih hematoloških parametara. Iako kod jedinki oba soja dolazi do povećanja broja neutrofilnih leukocita periferne krvi pri dozi od 2 mg L–1, promene u osnovnim proinflamatornim aktivnostima ovih ćelija su zapažene samo kod jedinki DA soja. Promene u broju neutrofilnih leukocita u krvi DA jedinki su praćene povećanjem broja granulocitnih prekursora u koštanoj srži, dok prisustvo neutrofila u plućima AO jedinki ukazuje na razmenu ćelija između periferne krvi i plućnog intravaskularnog pula ćelija. Diferencijalne sojno–zavisne promene u aktivnosti mononuklearnih ćelija periferne krvi su takođe zapažene. Razlike u efektu oralno unetog varfarina mogu da imaju implikacije za osobe na oralnoj varfarinskoj terapiji.Projekat ministarstva br. 17303

Strain differences in the toxicity of the vitamin K antagonist warfarin in rats

Warfarin (3-(α-acetonylbenzyl)-4-hydroxy coumarin) is a vitamin K (VK) antagonist that inhibits vitamin K-dependent (VKD) processes, such as blood coagulation. It also exerts an influence on some non-VKD-related activities. In this study, the effect of sub-acute (30-day) oral warfarin (2 and 1 mg L-1) intake on hematological parameters was examined in two rat strains, Albino Oxford (AO) and Dark Agouti (DA), that differ in their sensitivity to certain chemicals. Greater susceptibility to the anticoagulant effect of 2 mg L-1 of warfarin was observed in AO rats and was associated with an increase in the relevant hematological parameters in this strain. Although both strains responded to 2 mg L-1 of warfarin with quantitative changes in the peripheral blood leukocytes, differential bone marrow and lung responses were observed. Strain-related differences in the pro-inflammatory activity of peripheral blood granulocytes and in mononuclear cell IFN-γ production were observed. Recognition of differences in quantitative and qualitative effects of oral warfarin on processes other than hemostasis might be of relevance for those humans who are on warfarin therapy.Varfarin (3-α-acetonilbenzil)-4–hidroksikumarin) je antagonist vitamina K (VK) koji inhibira procese zavisne od ovog vitamina, uključujući koagulaciju krvi. Osim toga, on ispoljava i aktivnosti koje ne zavise od vitamina K kao što su anti-tumorska i imunomodulatorna aktivnost. U ovom radu je ispitan efekat subakutnog (30 dana) oralnog unosa varfarina na hematološke parametre i aktivnost leukocita periferne krvi kod dva soja pacova Albino Oxford (AO) i Dark Agouti (DA) koji se raz- likuju u osetljivosti na iste hemijske agense. Kod jedinki AO soja zapažena je veća smrtnost nakon konzumiranja doze od 4 mg L–1 kao i veća osetljivost na antikoagulantno dejstvo varfarina pri nižim dozama (2 mg L–1) koje je praćeno povećanjem nekih hematoloških parametara. Iako kod jedinki oba soja dolazi do povećanja broja neutrofilnih leukocita periferne krvi pri dozi od 2 mg L–1, promene u osnovnim proinflamatornim aktivnostima ovih ćelija su zapažene samo kod jedinki DA soja. Promene u broju neutrofilnih leukocita u krvi DA jedinki su praćene povećanjem broja granulocitnih prekursora u koštanoj srži, dok prisustvo neutrofila u plućima AO jedinki ukazuje na razmenu ćelija između periferne krvi i plućnog intravaskularnog pula ćelija. Diferencijalne sojno–zavisne promene u aktivnosti mononuklearnih ćelija periferne krvi su takođe zapažene. Razlike u efektu oralno unetog varfarina mogu da imaju implikacije za osobe na oralnoj varfarinskoj terapiji.Projekat ministarstva br. 17303

Intestinal and systematic immune effects of oral cadmium intake in rats

Kadmijum (Cd) je teški metal koji se nalazi u svim delovima životne sredine, nema poznatu biološku funkciju i ima štetno dejstvo na žive sisteme. Najčešći put izloženosti Cd je oralni, preko kontaminirane vode i hrane, kada je primarna meta toksičnosti ovog metala gastrointestinalni trakt. Poznato je da Cd oštećuje tkivo creva i remeti funkciju epitelne barijere koja je neophodna za održavanje imunske homeostaze, međutim mehanizmi imunotoksičnosti u ovoj regiji nisu dovoljno ispitani. Dodatno, poznato je da toksični efekti Cd mogu da zavise i od genetske osnove / soja eksperimentalnih životinja, ali sojne razlike u intestinalnoj toksičnosti oralnog unosa Cd do sada nisu ispitane. Ova disertacija je imala za cilj karakterizaciju efekta subhronične oralne primene Cd na imunski sistem creva pacova. Pacovi su bili 30 dana oralno (u vodi za piće) izloženi Cd u obliku kadmijum hlorida (CdCl2) u koncentraciji 5 ppm (5 mg Cd/l) i 50 ppm (50 mg Cd/l) Cd, što odgovara dozama prisutnim u životnoj sredini. U okviru lokalnog imunomodulatornog efekta Cd ispitivani su osnovni parametri imunskog odgovora u duodenumu (kao mestu najveće apsorpcije Cd) i mezenteričnim limfnim čvorovima (MLČ) koji dreniraju intestinum. U duodenumu su ispitani pokazatelji tkivnog oštećenja, oksidativnog stresa i zapaljenskih promena, a u MLČ su ispitane osnovne fenotpiske karakteristike i parametri aktivnosti ćelija ovog limfnog tkiva (celularnost, proliferacija, citokinski odgovor, urođeno-imunska aktivnost ćelija). Pored lokalnog, ispitan je i sistemski odgovor na oralni unos Cd uključujući humoralne i ćelijske parametre zapaljenske reakcije u krvi (promene hematoloških parametara, prisustvo medijatora inflamacije i oksidativnog stresa), kao i oksidativni stres i osnovne karakteristike urođenog i adaptivnog imunskog odgovora u slezini, limfnom organu u kome se uspostavlja imunski odgovor na antigene iz krvi...Cadmium (Cd) is a heavy metal which is found in every part of the environment, it does not have any known biological function and has an adverse effect upon the living systems. The most common way of Cd exposure is orally, through contaminated water and food, where the prime target of this metal toxicity is the gastrointestinal tract. It is known that Cd causes damage to intestinal tissue and disrupts the epithelial barrier function which is necessary for maintaining immune homeostasis, however, the immunotoxicity mechanisms in this region have not been examined sufficiently. Additionally, it is known that Cd toxic effects may depend also on genetic background / strain of experimental animals, but the strain-dependent differences in intestinal toxicity of oral Cd intake have not been examined to date. This dissertation was aimed at characterization of the effect of subchronic oral Cd administration on rat’s intestinal immune system. Rats were, for 30 days, orally (in drinking water) exposed to Cd in the form of cadmium chloride (CdCl2) at concentration of 5 ppm (5 mg Cd/l) and 50 ppm and (50 mg Cd/l) of Cd, which corresponds to the doses present in the environment. Within the local immunomodulatory Cd effect, basic parameters of immune response in duodenum (region of greatest Cd absorption) and in mesenteric lymph nodes (MLN) which drain intestine, were investigated. The indicators of tissue damage, oxidative stress and inflammatory changes were tested in duodenum, whereas in the MLN basic phenotype characteristics and parameters of this lymph tissue cells' activities (cellularity, proliferation, cytokine responses, and innate-immune cell activity) were tested. Besides the local, the systemic response to oral Cd intake was tested as well, including humoral and cellular parameters of the inflammatory reaction in blood (changes in hematological parameters, presence of inflammatory mediators and oxidative stress), as well as oxidative stress and basic characteristics of the innate and adaptive immune response in the spleen, a lymph organ in which immune response to blood borne antigens are generated..

Poly(DL-Lactide-co-ε-Caprolactone)/Poly(Acrylic Acid) Composite Implant for Controlled Delivery of Cationic Drugs.

Poly(DL-lactide-co-ε-caprolactone)/poly(acrylic acid) implantable composite reservoirs for cationic drugs are synthesized by sequentially applying photoirradiation and liquid phase inversion. The chemical composition and microstructure of reservoirs are characterized with Fourier transform infrared spectroscopy-attenuated total reflection (FTIR-ATR) and scanning electron microscopy (SEM), respectively. Drug loading and release properties are investigated using methylene blue as the drug model. Biocompatibility of reservoirs is examined through a series of in vitro tests and an in vivo experiment of subcutaneous implantation in Dark Agouti rats. Reservoirs show good ion-exchange capacity, high water content, and fast reversible swelling with retained geometry. Results of drug loading and release reveal excellent loading efficiency and diffusion-controlled release during 2 weeks. Biocompatibility tests in vitro demonstrate the lack of implant proinflammatory potential and hindered adhesion of L929 cells on the implant surface. Implants exhibit low acute toxicity and elicit a normal acute foreign body reaction that reaches the early stages of fibrous capsule formation after 7 days

Poly(DL-Lactide-co-ε-Caprolactone)/Poly(Acrylic Acid) Composite Implant for Controlled Delivery of Cationic Drugs.

Poly(DL-lactide-co-ε-caprolactone)/poly(acrylic acid) implantable composite reservoirs for cationic drugs are synthesized by sequentially applying photoirradiation and liquid phase inversion. The chemical composition and microstructure of reservoirs are characterized with Fourier transform infrared spectroscopy-attenuated total reflection (FTIR-ATR) and scanning electron microscopy (SEM), respectively. Drug loading and release properties are investigated using methylene blue as the drug model. Biocompatibility of reservoirs is examined through a series of in vitro tests and an in vivo experiment of subcutaneous implantation in Dark Agouti rats. Reservoirs show good ion-exchange capacity, high water content, and fast reversible swelling with retained geometry. Results of drug loading and release reveal excellent loading efficiency and diffusion-controlled release during 2 weeks. Biocompatibility tests in vitro demonstrate the lack of implant proinflammatory potential and hindered adhesion of L929 cells on the implant surface. Implants exhibit low acute toxicity and elicit a normal acute foreign body reaction that reaches the early stages of fibrous capsule formation after 7 days

Proinflammatory cytokine responses in skin and epidermal cells following epicutaneous administration of anticoagulant rodenticide warfarin in rats

Context: Dermal toxicity of coumarin anticoagulant rodenticides, such as warfarin, represents potential risk for workers handling these agents and for individuals applying easily available rodenticides in their households as well. Objective: In this study, proinflammatory effects of repeated epicutaneous administration of warfarin in rats were explored by examining inflammatory cytokine skin responses. Materials and methods: Ex vivo production of IL-1 beta, IL-6, TNF-alpha and IL-17 by skin explants and by epidermal cells isolated by enzyme (dispase/trypsin) digestion from skin repeatedly (once a day, three consecutive days) exposed to 10 mu g of warfarin was measured 24 h and 72 h following the last warfarin application by ELISAs for respective rat cytokines. Results: Warfarin treatment resulted in histological changes, but skin or epidermal cell viability were not compromised, judging by MTT reduction assay. Both skin and epidermal cells responded to administration of this agent by production of all examined inflammatory cytokines (skin explants by TNF-alpha and IL-17; epidermal cells by IL-1 beta and TNF-alpha) except IL-6. Discussion: Along with histomorphological changes, cytokines indicate functional consequences in treated skin. IL-1 beta production, that precede production of TNF-alpha, might be responsible for production of the latter cytokine. Sustained production of IL-1 beta suggests persistence of epidermal cell stimulation or existence of some amplification mechanisms. Requirements for T cells seem to exist concerning epidermal cell IL-17 production. Conclusion: Presented data provide additional new information concerning proinflammatory effects of warfarin.Ministry of Education, Science and Technological Development of the Republic of Serbia {[}173039

Warfarin affects acute inflammatory response induced by subcutaneous polyvinyl sponge implantation in rats

Purpose: Warfarin (WF) is an anticoagulant which also affects physiological processes other than hemostasis. Our previous investigations showed the effect of WF which gained access to the organism via skin on resting peripheral blood granulocytes. Based on these data, the aim of the present study was to examine whether WF could modulate the inflammatory processes as well. To this aim the effect of WF on the inflammatory response induced by subcutaneous sponge implantation in rats was examined. Materials and methods: Warfarin-soaked polyvinyl sponges (WF-sponges) were implanted subcutaneously and cell infiltration into sponges, the levels of nitric oxide (NO) and inflammatory cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6) production by sponge cells were measured as parameters of inflammation. T cell infiltration and cytokine interferon-γ (IFN-γ), interleukin-17 (IL-17) and interleukin-10 (IL-10) were measured at day 7 post implantation. Results: Warfarin exerted both stimulatory and suppressive effects depending on the parameter examined. Flow cytometry of cells recovered from sponges showed higher numbers of granulocytes (HIS48+ cells) at days 1 and 3 post implantation and CD11b+ cells at day 1 compared to control sponges. Cells from WF-sponges had an increased NO production (Griess reaction) at days 1 and 7. In contrast, lower levels of TNF (measured by ELISA) production by cells recovered from WF-soaked sponges were found in the early (day one) phase of reaction with unchanged levels at other time points. While IL-6 production by cells recovered from WF-soaked sponges was decreased at day 1, it was increased at day 7. Higher T cell numbers were noted in WF sponges at day 7 post implantation, and recovered cells produced more IFN-γ and IL-17, while IL-10 production remained unchanged. Conclusions: Warfarin affects some of the parameters of inflammatory reaction induced by subcutaneous polyvinyl sponge implantation. Differential (both stimulatory as well as inhibitory) effects of WF on inflammatory response to sponge implants might affect the course and/or duration of this reaction.Cutaneous and Ocular Toxicology (2017